Acanthamoeba Myosin I heavy chain kinase has been purified. It is a monomeric protein of molecular weight about 105,000 which phosphoryltes the heavy chains of both myosins IA an IB to a maximal extent of 1 phosphate/heavy chain and has no activity toward myosin II heavy chain. The K-binding of F-actin is the same for the enzymatically active phosphorylated myosin I and the enzymatically inactive unphosphorylated myosin I and is the same as the K-ATPase for the active enzyme (1 muM F-actin). The enzymatically more active, dephosphorylated from of Acanthamoeba myosin II has a higher K-ATPase for F-actin than does the much less active phosphorylated form of the enzyme so that its greater enzymatic activity is also attributable to its greater V-max. The dephosphorylated myosin II does have a lower K-binding for F-actin than does the phosphorylated form of the enzyme. The dephosphorylated enzyme also forms larger, more stable (toto KCl and MgATP) bipolar filaments than does the phosphorylated enzyme. Acanthamoeba calmodulin has been purified to homegeneity.